CA1238575A - Nucleic acid hybridization assay employing antibodies to intercalation complexes - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Nucleic acid hybridization assay methods and reagent systems for detecting a particular polynucleo-tide sequence in a test medium. An aggregate is formed in the assay reaction mixture comprising intercalation complexes between a nucleic acid intercalator and double stranded nucleic acid associated with the hy-bridization product of the sequence to be detected and a nucleic acid probe sequence. Hybridization of the probe with the sequence to be detected can then be determined by addition of an antibody, or a fragment thereof, capable of binding with the intercalation complexes in the formed aggregate and measuring the antibody or fragment thereof which becomes bound to such intercalation complexes associated with hybri-dized probe. In one preferred embodiment, this method eliminates the need to chemically modify the probe in order to form a labeled reagent. In another embodi-ment, the method provides an advantageous method for labeling the probe by chemical modification.

MS-1320-CIp-II

Description

ii7S-NUCLEIC ACID HYBRIDIZATION ASSAY

.

1. FIELD OF THE INVENTION
This invention relates to nucleic acid hy-bridization assay methods and reagent systems for detecting specific polynucleotide sequences. The principle of nucleic acid hybridization assays was developed by workers in the recombinant DNA field as a means for determining and isolating particular polynucleotide base sequences of interest~ It was found that single stranded nucleic acids, e.g., DNA
and RNA, such as obtained by denaturing their double stranded forms, will hybridize or recombine under appropriate conditions with complementary single stranded nucleic acids. By labeling such complementary probe nucleic acids with some readily detectable chemical group, it was then made possible to detect the presence of any polynucleotide sequence of interest in a test medium containing sample nucleic acids in s'ngle stranded form.

', , ~;~3~3S~
Z
In addition to the recombinant DNA field, the analytical hybridization technique can be appl;ed to the detection of polynucleotides of importance in ~he fields of human and veterinary medicine, agriculture, and food science, among others~ rn particular, the technique can be used to cletect an~ identify etiologi-cal agents such as bacteria an~ viruses, to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders such as sickle cell anemia and thalassemia and to detect cancerous cells.
A general review of the technique and its present and future signi~icance is provided in Biotechnology (August 1983), pp. 471-478.

2. DESCF~IPTION OF THE P~IOF~ A~T

The state-of-the-art nucleic acid hybridization assay techniques involve chemical modification of either the probe nucleic acid or sample nucleic acids for the purpose of labeling and detection. The necessity of chemically modifying nucleic acids severely limits the practical use of the technique since it requires the large-scale preparation of labeled probes involving complicated and expensive synthetic and purification procedures or the in situ synthesis of labeled sample nucleic acids by the analytical user. Tn particular, the resulting labeled polynucleotide must retain the ability to hybridize efficiently with its complementary sample or probe sequence. Such a requircment severely limits the availabilitY of useful synthetic approaches to label modification of polynucleotides intended for use in hybridization assays.

MS-1320-CIP~II

.. ;

~23~57~

The early hybridization techniques involved the use of raclioactive labels such as H, p, and I.
Labeled probes are synthesized enzymatically from radiolabeled nucleotides and a polynucleotide by such techniques as nick translation, end labeling, second strand synthesis, reverse transcription, and trans-cription. Thus, an additional requirement of such enzymatic methods is that the modified or labeled nucleotides must serve as effective substrates for the polymerase enzymes involved in the assembly of the labeled polynucleotide. Direct chemical modification of the polynucleotide is also possible, however, such a method is quite inefficient in incorporating labels into the polynucleotide and can affect the ability oE
the polynucleotide to undergo hybridization.
Because of the handling and storage disadvantages of radiolabeled materials, there has been considerable continuing efforts to develop useful nonradioisotopic labeling approaches. Such labels have included light emitting molecules such as fluorescers and chemilumin-escers, and ligand molecules which are capable of being specifically bound by counterpart binders which are in turn labeled with detectable chemical groups such as fluorescers and enzymes. Examples of ligand labels are haptens, which are specifically bound by antibodies, and other small molecules for which specific binding proteins exist, e.g., biotin which is bound by avidin.
Eritish Pat. No. 2,019,~08 describes polynucleotide probes which are labeled with biotin throuyh cytochrome C linlciny groups and which are then detectable by enzyme-labeled avidin. An alternative approach to labeliny probes with low molecular weight liyands such as biotin is described in European Pat. Appln.
63,879. In this technic~ue, 5-allylamine-deoxyuridine triphosphate (dUTP) derivatives are condensed with the desired ligand label and the thus modified necleo-tide is incorporated by standard enzymatic methods " ~

~ 5'7~
into the desired pr~be. The use of li~ht emitting labels is suggested by European Pat. Applns. 70,685 and 70,687. Other representatives of the l~atent literature pertaining to hybridization assays are U.S. Pat. Nos.
4,302,204 concerning the use of certain water solubLe polysaccharides to accelerate hybridization on a solid-phase; 4,358,535 concerning the detection of pathogens in clinical samples; and 4,395,486 concern-ing the detection of sickle cell anemia trait using a synthetic oligonucleotide probe.
Techniques for detecting directly the polynucleo-tide duplex formed as the product of hybridization between the sample and probe polynucleotides, and thereby dispensing with the chemical labeling of one or the other polynucleotide, have been generally un-successful. Attempts to generate antibodies which will selectively bind double str~nded DNA/DNA hybrids over single stranded DNA have failed [Parker and Halloran, "Nucleic Acids in Immunology", ed. Plescia and Braun, Springer-Verlag, NY(1969) pp. 18 et seq].
Some success has been achieved in generating anti!-odies that will bind RNA/DNA mixed hybrids and have low affinity for the single stranded polynucleotides [Rudkin and Stollar, Nature 265:472~1977); Stuart et al, PNAS(USA)78:3751(1~81); Reddy and Sofer, Biochem.
Biophys. Res. Commun. 103:959(1981); and Nakazato, Biochem. 19:2835(1980)l, however, the sensitivity of these methods has not reached the levels required for clinical hybridization tests and one would have to use ~NA ~robes which are well known to be quite unstablc.
Accordingly, there is an established need ~or a technique for detecting hybridization without requir-ing chemical modiEication of polynucleotides or in-volving a labeling method of relative simplicity.Further, such technique should enable the use of a !

.. .

.. ~

~ 3~S~S

variety of labels, particularly of the nonradioisotopic type. A nucleic acicl hybridization assay method and reagent system having these and other advanta~es are principal objectives of the present invention.
U.S. Pat. No. 4,257,774 describes a method for detecting various compounds that interact with nucleic acids, particularly compounds sllspected as possible mutagens or carcinogens~ l)y measuring the ability of such compounds to inhibit the binding of intercalators such as acridine orange to nucleic acids. Poirier, M.C. et al (1982) PNAS 79:6443-6447 describe the preparation of a monoclonal an~ibody selective for certain c~is-platinum/double stranded DNA complexes over the free cis-platinum compound and double stranded DNA.
SUMMARY OF THE INVENTION
It has now been found that hybridization which occurs between sample nucleic acid and the probe in nucleic acid hybridization assays can be detected ad-vantageously by means of an antibody, or an appro-priate binding fragment thereof, capable of binding with intercalation complexes formed in association with hybridized probe. In essence, a particular polynucleotide sequence is detected in a test medium containing single stranded nucleic acids by forming a hybriclization aggregate or product compris-ing hybridized probe and a nucleic acid intercalator compound bound to double stranded nucleic acid in the form of intercalation complexes. 'lhe antibody or fragment thereof is then used to detect intercalation complexes in the hybridization aggregat^.
The use of nucleic acid hybridization as an analy-tical tool is based fundamentally on the double stranded, duplex structure of DN,~. The hyclrogen bonds ,

3~;75 between the purine ancl pyrimidinc! bases of the resp~c-tive strands in double stranded DNA can be reversibly broken. The two complelllentary single strands of DNA
resulting from this mcltin~ or dcnaturation of DNA
will associate (also referred to as reannealing or hybridization) to reform the duplexed structure. As is now well known in the art, contact of a first single stranded nucleic acicl, either DNA or RNA, which comprises a base sequence s~lfficiently comple-mentary to (i.e., "homolo~ous with") a second s;n~lestranded nucleic acid under appropriate so'ution condi-tions, will result in the formation oE DNA/D~A, R~A/DNA, or RNA/RNA hybrids, as ~he case may be.
The present invention enables the detection of formed hybrids by inducing an immunogenic modification of double stranded nucleic acid in the region of hybridization or in flanking regions. The resulting product can then be detected by conventional assay schemes based on the binding of specific antibody to the epitopes or antigenic determinants formed on the hybridization product. Thc requisite immunogenic modification of double stranded nucleic acid is accom-plished principally by binding o~ a molecule, usually a low molecular weig}lt compound, to the duplex. Such binding results in the creation of an antigenic deter-minant which distinguishes double stranded nucleic acid from both single strancled nucleic acid and the free, unbound modiE:ier molecule. Preferably, this is accomplished by employing a modifier compound which is 3G essentially incapable o binding Wit}l single stranded nucleic acid and which forms a binding complex with double stranded nucleic acid which alters the normal helical relationship o~ the complementary strands oE
the duplex, Ms-l32o-cIp-II

3L23~5~5 Such modifier molecul~ as describe~l herein is a nucleic acid intercalator which prereren~ially will interact with the normal nucleic acid helix by a non-covalent insertion bet~een base pairs. Such insertion causes, in this preferr~d interaction, the tertiary structure of the helix to change by unwin~ing and elonga-tion along the helical ~is. ~ scllematic representation of this preferred intercal<ltion interaction is shown in Fig. 1 of the drawin~s. T]le resultin~ terc~lation complex is characteri7ed by newly rorme~l arlti~enic ~e-terminants which are understoocl to comprise th~ inter-calated modifier compound and the reo7-iented phosohodi-esterase backbones of the respective strands of the duplex.
Preferably, the intercalator compound is one ol the generally planar, aromatic organic molecules known to form intercalation complexes with double stran~ecl nucleic acid. Such compounds are exemplified by the acridine dyes, e.g., acridine orange, the phenanthri-dines, e.g., ethidium, the phenazines, rurocounlarins, phenothiazines, quinolines, and the like as are more fully described below. It should be clearly understood that while the present invention will be hereinafter described with particular reterence to such intercala-tor compounds, the present invention contemplates theuse of equivalent modifier molecules which, as des-cribed above, will bind to double stranded nucleic acid to induce an immunogenic change in the duplex.
In accordance with the present invention, the intercalator can be combine~d with the test medium, and thereby become expose-l to the double stranded nucleic acids present and/or ~orming in the hybriclizatio~ eac-tion mixture, as a separate, ~ree compound and bind noncovalently to such double stranded nucleic aci~s to form intercalation complexes. Altern.ltively, thc intercalator can be appropriately linked by chemical ,, MS-1320-CIP~II
"

ii75 bonds, preferably covalent bonds, to tlle l~robe. ~n the former case, the present invention provides a method for performing a hybridization assay without the need to chemically mo~i~y either the sample or probe polynucleotide in order to ~etect hybri~iz~tion.
In the latter case, a simple, syntlletically straigllt-forward means for lal~eling polynucleotides or the hybridization aggregate is provi~e(l by ~he use of photoreactable forms o~; tlle inter~-~lator.
In all embodiments, tlle present inven-tion pro-vides a highly versatile, sensitive, and specific method for detecting hybridization based on antibody binding t~ the intercalation complexes in the aggre-gate formed. Of course, appropriate fragments and polyfunctional forms o~ the antibody can be used as described more fully below, and it will be understoocL
that when used in this disclosure tlle term antibo(ly will mean its fragmented ancl polyfunctional forms as well, unless otherwise notecl. Determining the binding of antibody to intercalation complexes can be accom-plished in a variety of conventional manners and preferably involves the use of antibody labelecL with a detectable chemical group such as an enzymatically active group, a fluoresccr, a luminescer, a specifically Z5 bindable ligand, or a radioisotope.
The invention is applicable to all conventional hybridization assay formats, ancL in general to any format that is possible based on formation of a hybridi-zation product or aggle~ate comprising double strancled nucleic acid. In particular, the unicl~e cLetection schemc of the present invention can be used in solu-tion and solid-phase hybridization formats, including, in the latter case, formats involving immobilization of either sample or probe nucleic acids and sandwich formats.

~23~ 75 g The hybridization product or aggregate formed according to the present invention comprises hybridize~
probe and intercalator boun~l to double strancled nucleic acid in the form of intercalation complexes. The intercalation complexes can involve ~louble stranded regions formed by hybridiz;ltion be~ween sample an~
probe nucleic acids. ,~lterna~ively, such double stranded regions can bc coml-rised in the probe itself and in such case can a~ditionally be intercalated prior to use of the probe in the assay. Thus, the detectable intercalation complexes can be formed in sit~ during the assay or can be existent in the pro~e reagent as presented to the test medium. Further, the intercalation complexes can be chemically linked to one or both of the strands of the intercalated duple.Y.
In general, any variation can be -rollowed provided that the hybridization product ultima+ely comprise inter-calation complexes detectable by tlle antibody binding phenomenon which is the underlying hasis o-f the present invention.
Thus, the present invention provides an advan-tageous nucleic acid hybridization method and reagellt system. Additionally, there is provided a novel antibody reagent capable o-f binding with intercalatio complexes. Furthermore, besides the cletection of particular polynucleotide ~cquences, the present invention provides a general methocl for cletecting double stranded nucleic acid l)y aclding intercalator and tlle anti-(intercalation complex) antibody and cleterminin~
antibody binding.

~IS-1320-CIP-II

~ ~3~ 5 The advantages of the present invention are ~igni-ficant and many. The invention is amenable ~o a wide variety of nonradioactive detection methods. Further~
labeling of nucleic acids is straigh~forward and uses easily synthesized reagents. Labeling with the inter-calator does not require exnensive polymerases, and the labeling density of the intercalator can be easily controlled. Certain preferred embo~iments have other advantages. In those embodiments in which the intercalator-nucleic acid comple~ is for~ed in ~itl~, no prior synthesis of the complex is required and this approach can be used in a format in which a probe is immobilized on a solid support and immersed in a solution containing the specimen nucleic acid. In the embodiment where the intercalator is covalently coupled to the nucleic acid, the intercalator is attached to the probe during the manufacturing process, resulting in a controlled level of saturation. Tllis approach also minimizes user exposure to an inter-calating agent, many of which may be potentiallyhazardous.

BRIEF DESCRIPTION OF Tl-IE DRAWINGS

Fig. l, as described above, is a schematic representation of the preferred interaction between intercalator and double stranded nucleic acid which results in an intercalation complex that is detectable by antibody.
Fi~s. 2-i are schematic diagrams of four preferred hybridization formats for use in the present invention.

Ms-l32o~cIp-II

~.~3~S~;

DESCRIPTION OF I`IIE PREFERRED EMBODIMENTS

Int~ cal.ator As described above, the intercalator compound preferably is a low molecular weight, planar, usually aromatic but sometimes polycyclic, molecule capable of binding with double stranded nucleic acids, e.g., DNA/DNA, DNA/RNA, or RNA/RNA duplexes, usually by insertion between base pairs. The primary binding mechanism will usually be noncovalent, with covalent binding occuring as a second step where the intercala-tor has reactive or activatable chemical groups which will form covalent bonds with neighboring chemical groups on one or both of the intercalated duplex strands. The result of intercalation is the spreading of adjacent base pairs to about twice their normal separation distance, leading to an increase in molecular length of the duplex. Further, unwinding of the double helix of about 12 to 36 degrees must occur in order to accomodate the intercalator. Ceneral reviews and further information can be obtained from Lerman, J. Mol. Biol. 3:18(1961); Bloomfield et al, "Physical Chemistry of Nucleic Acids", Chapter 7, pp. 429-476, Harper and Rowe, NY(1974); Waring, Nature 219:1320 ~1968); Hartmann et al, Angew. Chem., ~ngl.
25 Ed. 7:693(1968); Lippard, Accts. Chem. Res. 11:211~1978);
Wilson, Intercalation Chemistrytl982),445; and Berman et al~ Ann. Rev. Bio~h~rs. Bioen~. 10:87(1981).

.. ..
. , A wide variety of intercalating agents can be used in the present invention. Some classes of these agents and examples of specific compounds are given in the following table:

Intercalator Classes Literature References and Representative Compounds . _ A. Acridine dyes Lerman, sup~; Bloom-field et al, supra;
proflavin, acridine orange, Miller et al, Bio-quinacrine, acriflavine polymers 19:2091(1980) B. Phenanthridines Bloomfield et al, supra;
Miller et al, supra ethidium coralyne Wilson et al, J. ~ed.
Chem. 19:1261(1976) ellipticine, ellipticine Festy et al, FEBS
cation and derivatives Letters 17:321~1971);
Kohn et al, Cancer Res.
35:71~1976); LePecq et al, PNAS ~USA)71:
5078~1974~; Pelaprat et al, J. Med. Chem.
23:1330(1980) C. Phenazines Bloomfield et al, supra 5-methylphenazine cation D. Phenothiazines ibid chlopromazine E. Quinolines ibid chloroquine quinine F. Aflatoxin ibid MS-1320-CIp-II

:

3~75 G. Polycyclic hydrocarbons ibid and their oxirane derivatives 3,4-benzpyrene benzopyrene diol Yang et al, Biochem.
epoxide, l-pyrenyl- Biophys. Res. Comm.
oxirane 82:929(1978) benzanthracene-5,6-oxi~e Amea e~ al, Science 176:47(1972) 10 H. Actinomycens Bloomfield et al, supra actinomycin D

I. Anthracyclinones ibid ~-rhodomycin A
daunamycin 15 J. Thiaxanthenones ibid miracil D

K. Anthramycin ibid L. Mitomycin Ogawa et al, Nucl.
Acids Res., Spec.
Publ. 3:79~1977);
Akhtar et al, Can. J.
Chem. 53:2891~1975) M. Platinium Complexes Lippard, supra N. Polyintercalators echinomycin Waring et al, Nature 252:653~1974);
Wakelin, Biochem. J.
157:721(1976) quinomycin Lee et al, Biochem. J.
triostin 173:115(1978); Huang BBM928A et al, Biochem. 19:
tandem 5537(1980), Viswamitra et al, Nature 289:
~17~1981) ~23~7S

diacridines llePecq et al, PNAS
~U~)72:2915(1975);
Carrellakis et al, Biochem. Biophys.
Ac-ta 418:277(1976);
Wakelin et al, Bio-cllem 17:5057(1978);
Wakelin et al, ~EB~S
Lett. 104:261(1979);
Capelle et al, ~io-chem. 18:3354(1979!;
Wright et al, 13iochem.
19:5825(1980); ~ernier et al, Biochem. J.
1 199:479~1981); King et al, Biochem. 21:
4982(19~2) ethidium dimer (;augain et al, Bio-chem. 17:5078(1978);
Kuhlman et al, Nucl.
Acids Res. 5:2629 (1978); Marlcovits et al, Anal. Biochem.
94:259(1979); Dervan et al, JACS 100:1968 (1978); ibid 101:
3664(1979).
ellipticene dimcrs Debarre et al, Compt.
and analogs Rend. Ser. D 284:
8l(1977); Pelaprat et al, J. Med. Chem.
23:1336(1980) heterodimers Cain et al, J. Med.
Chem. 21:658(1978);
Gaugain et al, Bio-chem. 17:5078(1978) trimers llclnsen et al, JCS
Chem. Comm. 162tl983);
Atnell et al, JACS 105:
2913(1983) O. Norphillin A l.oun et al, JACS 104:
3213(l982) ' ' ~, ~ ~ .

, .- , ~2~ S

P. Fluorenes and fluorenones ~loomfield et al, supra fluorenodiamines Wltkowskl et al, Wiss. Beitr.-Martin-Luthêr-Univ. Halle Wittenberg, 11(1981) Q. Furocoumarins angelicin Venema et al, MCG, Mol. Gen. Genet.
179;1 (1980)

4,5'-dimethylangelicin Vedaldi et al, Chem.-Biol. Interact. 36:
275(1981) psoralen Marciani et al, 7.
Naturforsch B 27(2):
196(1972) 8-methoxypsoralen Belo~nzov et al, Mutat.
Res. 84:11(1981);
Scott et al, ~hotochem.
Photobiol. 34:63(1981)

5-aminomethyl-8- l~ansen et al, Tet. Lett.
methoxypsoralen 22:1847~1981) 4,5,8-trimethylpsoralen Ben-}lur et al, Biochem. Biophys.
Acta 331:181(1973) 4'-aminomethyl-4,5,8- Issacs et al, Biochem.
trimethylpsoralen 16:1058(1977) xanthotoxin Hradecma et al, Acta Virol. (Engl. Ed.) Z6:305(198~) khellin Beaumont et al, Biochim. Biophys.
Acta 608:1829~1980) R. Benzodipyrones Murx et al, J. Het.
Chem. 12:417(1975);
Horter et al, Photo-chem. Photobiol. 20:
` 407(1974) S. Monostral Fast Blue Juarranz et al, Acta Histochem. 70:130 (1982) ' ~

.
~ .

123~

Several embodiments of the prescnt invention in-volve the chemical, e.g., covalent, linka~e of the intercalator to one or both of the complementary strands of a duplex. Essentially any convenient method can be used to accomplish such lin~age. Co~veniently, the linkage is formed by ef~ecting intercalation with a reactive, preferably pllotoreactivc intercalator, followed by the linking reaction. A particularly useful method involves the use of azidointercalators.
The reacti~e nitrenes are readily ~enerated at long wavelength ultraviolet or visible li~ht and the ni-trenes of arylazides prefer insertion reactions over their rearrangement products [see White et al, Methods in Enzymol. 46:644(1977)]. Representative azidoin-tercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazide, ethidium diazide, ethidium dimer azide [Mitchell et al, JACS 104:~265(1982)], 4-azido-7-chloroquinoline, and 2-azidofluorene. Other useful photoreactable intercalators are the furocoumarins which form [2~2] cycloadducts with pyrimidine residues.
Alkylating agents can also be used such as bis-chloroethylamines and epoxides or aziri~ines, e.g., a~latoxins, polycyclic hydrocarbon epoxides, mitomycin, and norphillin A.
Depending on the hybridization format involved, as will be described in detail below, chemically linked intercalation complexes can be used in a variety o~ manners in the present invention. They can be formed in situ in the hybridization reàction mixt~lre or in a process step thereafter, cr can be a step in the synthesis of a labeled probe or sample nucleic acid.
In the latter case, where intercalation occurs in the region o~ complementarity between the probe and sample nucleic acids~ mono-linkages will be accomplished MS-1320-CIP-~

, ~ L~3i:3575 followed by denaturing oF ~uch region to yield single stranded nucleic acid with chemically linked inter-calator oriented such that upon hybridization, the linked intercalator will assume an intercalation position.

HYBRI DI ZA TI ON FORMA ~v' .4 ND PROBES

The probe will compri~e at least one single stranded base sequence substantially complementary to or homologous with the sequence to be detected.
However, such base sequence need not be a single con-tinuous polynucleotide segment, but can be comprised of two or more individual segments interrupted by nonhomologous sequences. These nonhomologous sequences can be linear, or they can be self-complementary and form hairpin loops. In addition, the homologous region of the probe can be flanked at the 3'- and 5'-terminii by nonhomologous sequences, such as those comprising the DNA or RNA of a yector into which the homologoùs sequence had been inserted for propagation.
In either instance, the probe as presented as an analy-tical reagent will exhibit detectable hybridization at one or more points with sample nucleic acids of interest. Linear or circular single stranded poly-nucleotides can be used as the probe element, with major or minor portions being duplexed with a com-plementary polynucleotide strand or strands, provided that the critical homologous segment or segments are in single stranded orm and available for hybridlza-tion with sample DNA or RNA. i'articularly preferred will be linear or circular probes wherein the homo-logous probe sequence is in essentially only single stranded form [see particularly, Hu and Messing, Gene 17:271-277(1982~].

MS-1320~CIP-II

'75 Where the probe is used in a hybridization forma~
calling for use of an intercalator-labeled probe, as will be seen below,such probe can ~e in a variety of forms such as a completely sin~le stranded polynucleo-tide having intercalator chemically linked theretowhereby hybridization results in formation of inter-calation complexes. Alternatively, the probe can com-prise a double stran~ed portion Ol portions which have been intercalated, optionally with covalent linkage of the intercalator to one or botll strands in the duplex.
In terms of hybridization formats, the present invention is focused on formation of a hybridization aggregate comprising the hybridized probe and the inter-calator bound to duplexes in the form of the antibody-detectable intercalation co~plexes. Thus, the eventof hybridization is associated with the formation of the detectable intercalation complexes. Fundamentally, the resulting intercalation complexes in the aggregate can be in the region of hybridization between the sample and probe nucleic acids or can be in a double stranded region remote from the hybridi ation region.
In such latter case, the intercalated re~ion can be formed during the assay or can be in the intercalated state when brought to the assay, e.g., covalently linked or noncovalently intercalated double stranded regions serving as labels Eor the probe.
Practice of the present analytical method is not limited to any particular hybridization format. Any conventional hybridization technique can be used. As improvements are made and as eonceptually new formats are developed, such can be readily applied to carrying out the present method. Conventional hybridization formats which are particularly useful include those wherein the sample nucleic acicls or the polynucleotide probe is immobilized on a solid support (solid-phase hybridization) and those wherein the polynucleotide MSl320-cIP-II

~3~35;7~

species are all in solution (solution hybridiz~tion).
In solid-phase hybridization formats, one o~
the polynucleotide species participating in hybridiza-tion is fixed in an appropriate manner in its single stranded form to a solid support. Useful solid supports are well known in ~he art and include those which bind nucleic acids cither covalen~ly or non-covalently. Noncovalent supports which are generally understood to involve hydrophobic bonding include naturally occurring and synthetic polymeric materials, such as nitrocellulose, derivatized nylon, and fluorinated polyhydrocarbons, in a variety of forms such as ~ilters or solid sheets. Covalent binding supports are also useful and comprise materials hav-ing chemically reactive groups or groups, such asdichlorotriazine, diazobenzyloxymethyl, and the like, which can be activated for binding to polynucleotides.
A typical solid-phase hybridization technique begins with immobilization of sample nucleic acids onto the support in single stranded form. This initial step essentially prevents reannealing of complementary strands from the sample and can be used as a means for concentrating sample material on the support for en-hanced detectability. The polynucleotide probe is then ~5 contacted with the support and hybridization detected by antibody binding as desc-~ibed herein. The solid support provides a convenient means ~or separating antibody which binds to intercalation complexes associated with hybridized probe ~rom that which does not so bind.
Another m~thod o~ interest is the sandwich hy-bridi~ation technique wherein one of two mutually ex-clusive fragments of the homologous sequence of the probe is immobilized and the other is labeled. The presence of the polynucleo~ide sequence of interest Ms-l32o-cI~
, ~3~

results in dual hybridizatio]l to the immobilized and labeled probe segments, again with the same ulti-mate measurement o~ support-associatc~l intercalation complexes. See Metho~s in Enzymology 65:46~(1980) and Gene 21:77-85~1983) for ~urther details.
For purposes of better illustration, the ~ollow-ing solid-phase hybridization metho~s are particularly useful in the present inve]ltion. Scllematic diagraTns of these basic methods are provided in the drawings.

Method type 1 In this method, illustrated in Fig. 2, the single stranded nucleic acids from the liquid test medium are first immobilized on a solid support. A hybridiza-tion reaction mixture is then formed by contacting the immobilized sample nucleic acids (S) with the probe (P) which in this case comprises, in addition to the com-plementary single stranded portion, at least one double stranded portion which is chemically linked with the intercalator (I) in the form of intercalation complexes.
A particularly useful form of the probe is the circu-lar form described by ~lu and Messing, supra. The resulting hybridization aggregate comprises the immobilized polynucleotide of interest hybridized with the probe which has a covalently linked, inter-calated double stranded region. The solid supportcarrying immobilized duplexes is then preferentially separated from the remainder of the reaction mixture.
The antibody (Ab) is added, pre~erably labeled with a detectable group, and the resulting immobilized anti-body bound to intercalation complexes in the aggregatcis sepa~ated from the remainder of the reaction mixture.
The antibody bound to the support is then determined to complete the assay. Alternatively, the antibody in the separated solution can be determined; although this will generally be less preferred since a large excess of ` antibody is normally used.
MS-1320-CIp-II

~3~7~i A variation of this mcthod is to employ a probe such as above, but not ha~rin~ covalently linke~ in~cr-calator bound to the double strande~ rcgion. Rather, the intercalatoIt is a~ded to the im1nobilized ~ggregate resulting in the ~ormation of intcrcalator complexes in both the double stran~e~ portion Or the probe an~ the duplexed region forme~ by hybridization.

Method type 2 This is a sandwich format and is illus~rate~ in Fig. 3. A reaction mixture is ~ormed among the test medium containing the sequence o~ interest (S) and the first and second probes, each comprising respectively at least one base sequence complementary to a mutually exclusive portion of the sequence Or interest. The first probe (Pl) is immobilized on a solid support and the second probe (P2) is labeled with covalently linked, intercalation complexes as in Method type 1 above. The resulting hybridization aggregate comprises the sequence of interest hybridized to both the immobilized first probe and the intercalation complex-labeled second probe. The antibody is added, prefer-ably in labeled form, and the resulting immobilized antibody bound to intercalation complexes in the aggregate is separated Erom thc remainder of the reac-tion mixture. The bound antibody is ~etermined tothen complete the assay.
There are several useful variations oE this method. First, as in the casc O r thc variation of Method type 1, one can employ a probe which does not comprise covalently linked intercalator, but rather can add free intercalator to the immobilized aggre-gate resulting in the ~ormation of intercal~tor com-plexes with all available double stranded regions.
Also, as an alternativc to using a second probe with ~3~S

a double stranded portion, one can llse a probe of entirely single stranded nucleic acid with intercalator chemically linked thereto so that upon hybridization there are for-ned interealation complexes, or with intercalator being a~dcd so that intercalation occurs between the duplexes rormed betwecn thc two probes and the sequence to be detected.

Method type 3 Fig. 4 illustrates a ~urther preferred soli~-phase format. The sample nucleic aci~s are contacte~
with immobilized probe and preferably the resulti~g immobilized duplexes are separated From the remainder of the reaction mixture. In this format, the probe is in single stranded form. The resulting hybridiza-tion product comprises the immobilized probe hybridized with the sequence of interest. Also, this format allows significant reannealing between complemen~ary regions of sample nucleic acid which can take place on the immobilized aggregate. Such reannealing works to the advantage of the assay since it provides addi-tional double stranded nucleic acid ~or subsequent intercalation. The next step in the assay is to add intercalator and the antibody, again pre~erably in a làbeled form. The assay is completed by separation and antibody determination steps as in the previous formats.

Method type 4 In this method, illustrated in ~'ig. S, the single stranded sample nucleic acids are contacted with immobilized probe where, in this case, such probe is MS-1320-C~

., , .

~3~5~75 chemically linked, e.g., covalently, to the intercalator such ~hat duplex formation in the region of the linked intercalator results in formation of intercalation complexes. This is a highly advantageous format in that it is the only known technique wherein the probe is both immobilized and labeled, requiring no immobilization or labeling step to be performed at the time of the assay. The resulting aggregate comprises covalently linked, intercalation complexes in the region of hybridization between sample and probe nucleic acids and in any reannealed sample regions. Antibody is-than added and the assay completed as in the previous formats.
This format provides the advantage of eliminating the need for the analyst to handle solutions of the free intercalator which in some cases can be potentially hazardous. A simple variation of this technique is to immobilize sample nucleic acids 2Q rather than the labeled probe and proceed in the normal fashion. This is somewhat less advantageous but is a practical assay approach.
A variety of solution-phase hybridization ormats can also be applied to the present invention. Such formats are characterized by the feature that the hybridization step involves soluble forms of both the sample nucleic acids and the probe. This can result in significantly faster hybridizations since the kinetics are much faster when both strands are in solution compared to when on is immobilized. Normally, subsequent to the hybridization step, the resulting hybrids are rendered immobile for purposes of detection~ Such immobilization can be accomplished in a variety of ~231~S75 ways. Conventionally it is known to selectively immobilize cuplexes by exposure to adsorbents such as hydroxyapatite and nitrocellulose membranes.
A par~icularly useful approach to immobili~ing hybrids ~ormed from a solution-phase hybridization involves the use of a probe which comprises a binding site for a binding substance. After the hybridization step then, one can add an immobilized form of the binding substance which will effectively bind and immobilize the hybrids through the binding site on the probe. Such binding site can be present in a single stranded hybridizable portion of the probe or can be present as a result of a chemical modification of the probe. Examples of binding sites existing in the nucleotide sequence are where the probe comprises a promoter sequence (e.g., lac-promoter, trp-promoter) which is bindable by a promoter protein (e.g., bacteriophage promoters, RNA polymerase), or comprises an operator sequence (e.g., lac operator) which is bindable by a repressor protein (e.g., lac repressor), or comprises rare, antigenic nucleotides or sequences (e.g., 5-bromo or 5-iododeoxyuridine, Z-DNA) which are bindable by specific antibodies [see also British Pat. Spec.
2,125,964]. Binding sites introduced by chemical modification of the probe are particularly useful and normally involve linking one member of a speci~ic binding pair to the probe nucleic acid.
Useful binaing pairs from which to choose include biotin/avidin, haptens and antigens/antibodies, MS-1320 CIP-II carbohydrates/lectins, enzymes/inhibitors, and the like. Where the binding pair consists of a proteinaceous member and a nonproteinaceous member, it will be preferred to ~3~ S

llnk the nonproteinaceous member to the probe since the proteinaceous member may be unstable under the denaturing conditions of hybridization of the probe. Preferable systems involve linking the probe with biotin or a hapten and employing immobilized avidin or anti-hapten antibody, respectively. Preparation of useful ligand-labeled probes is known in the literature [Langer et al (1981) Proc. Natl. Acad. Sci. 7~:6633; Broker (1978) Nucl. Acids Res. 5:363; Sodja et al (1978) Nucl. Acids Res. 5:385; Tchen et al (1984) Proc.
Na-tl. Acad. Sci. 81:3466]. Immobilization of the binding substane can fol~ow conventional techniques.
A large variety of methods are known ~or immobilizing proteins on solid supports and these methods are applicable to the immobilization of the binding substance ~see Methods in Enzymology, Vol.
44(1976)]. Antibodies, for example, are immobilized either by covalent coupling or by noncovalent adsorption. Noncovalent methods frequently employed are adsorption to polystyrene beads or microparticles and to polyvinylchloride surface. Many covalent methods are used for immobilizing proteins and a few include cyanogen bromide activated agaroses and dextrans;
glutaraldehyde activated nylons and polyacrylamides: and epoxides on acrylic and other supports.

~IL23~ 75 The a~ove illustrativP methods are particularly preferred, however, the present invention is not limited to any particular ~ybridization format. Any approach to an assay can be followed provided that detecta~le intercalation complexes result in associa-tion with hybridization of the probe nucleic acid.
For instance, in addition to the above methods, one can devise a solution phase hybridization format wherein a solid-phase antibody to intercalation complexes is employed to immobilize hybridized probe. There will be suficient intercalation complexes formed in the hybridization product between sample and probe nucleic acids, the latter being in esscntially only single stranded form, such that both solid-phase antibody and labele~ antibody can bind. The amount of label associated with the solid-pllase is then measured and is related to the presence of the sequence to be determined. Other useful formats will be evident to one of ordinary skill in the art.

;

.
. ., , ~, ~, ..... ... ,:

~3B~

ANTIBODY REAGENT A~D DETEC~ION SCHE~ES

A fundamental principle of the present invention is the ability to first bind an antibody, or a fragment or some other equivalent thereof, to the hybridization aggregate comprising hybridized probe and then to detect such antibody binding. As s~ated above, the antibody reagent can consist of whole antibodies, antibody fragments, polyfunctional antibody aggre~ates, or in general any substance comprising one or more 10 intercalation complex-specific binding sit~es from an antibody. When in the form of whole antibody, it can belong to any of the classes and subclasses of known immunoglobulines, e.g., IgG, IgM, and so forth. Any fragment of any such antibody which retains specific 15 binding affinity for intercalation complexes can also be employed, for instance, the fragments of IgG con-ventionally ~nown as Fab, F(ab'), and F~ab')2. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where 20 appropriate.

M~132 a~ P-~I
,;

:' '' ..... , ~ "
~, . . : -, .... .

2~
The immunoglobulin source COI th~ antibo~y rea-gent can be obtainecl in any available manner such as conventional antiserum and monoclonal techniques.
Antiserum can be obtained by well-established ~echni-ques involving immunization of an .lnimal, such as amouse, rabbit, guinea pig or goat, Wit]l an appropriate immunogen. The immunogen will usually comprise an ionic complex between a cationic protein or protein derivative (e.g., methylated bovine serum albumin) and the anionic intercalator-nucleic acid complex.
Ideally, the intercalator should be covalently coupled to the double stranded nucleic acid. Alternatively, the intercalator-DNA conju~ate can be covalently coupled to a carrier protein. The immunoglobulins can also be obtained by somatic cell hybridization techniques, such resulting in what are commonly re-ferred to as monoclonal antibodies. The immunogen used for primary injections leading to hybridoma formation will be as described above.
The antibody reagent will be characterized by its ability to bind with an intercalation complex formed between a selected intercalator and double stranded nucleic acid in general without regard to the specific base sequences proximate to the site of intercalation.
Furthermore, it will be substantially incapable oE
binding to single strande~ nucleic acids or to free intercalator. As a result, antibody binding will occur only at intercalation complexes which by proper design of the assay format will be signiEicantly present only in association with hybridized probe.

The binding of the antibody reagent to the hybridization aggregate in the present method can be detected by any convenien~ technique. Advan-tageously, the antibody reagent will itself be labeled with a detectable chemical group. Such detectable chemical group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassàys and in general most any label useful in such methods can be applied to the present invention. Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem.(1976)22:1243), enzyme substrates (see British Pat. Spec. 1,5489741), coenzymes (see U.S.
Pat. Nos. 4,230,797 and ~,238,565), and enzyme inhibi-tors (see U.S. Pat. No. 4,134,792); fluorescers ~see Clin. Chem.(1979)25:353); chromophores; luminescers such as chemiluminescers and bioluminescers (see (Clin. Chem.(1979)25:512, and ibid, 1531); specifically bindable ligands; proximal interacting pairs; and radioisotope5 such as 3H 35S 32p 125I and 14C
Such labels and labeling pairs are detected on the basis of their own physical properties ~e.g., fluores-cers, chromophores and radioisotopes) or their reac-tive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors). For example, a cofactor-labeled antibody can be detected by adding the enzyme or which the label is a cofactor and a substrate for the enzyme. A hapten or ligand (e.g., biotin) labeled antibody can be detected by adding an anti- -body to the hapten or a protein (e.g., avidin) which binds the ligand, tagged with a detectable molecule.
Such detectable molecule can be some molecule with a measurable physical property ~e.g., fluorescence or absorbance) or a participant in an enzyme reaction 2 C - C T ? ~I. I: .

.

.
. ~ . . .
. ' ~ , .

~3~S7Si (e.g., see above list). For example, one can use an enzyme which acts upon a substrate ~o generate a product with a measurable physical property. Examples of the latter include, but are not limited to, ~-galactosidase, alkaline phosphatase and peroxidase.
For in situ hybridization studies, ideally the final product is water insoluble. Proximal interacting or linking labels as are known in the immunoassay field ~see Clin. Chem. 27:1797~1981) and U.S. Pat. Nos.
3,996,345 and 4,233,402) can be applied to the pre-sent method by using two different populations of antibodies, one labeled with one member of the pair and the other labeled with the other of the pair.
For instance, a first portion of antibodies to inter-calation complexes is labeled with a fluorescer and a second portion is labeled with a quencher. The presence of intercalation complexes is indicated by quenching of fluorescence due to the proximate bind-ing of first and second portion antibodies along the ~0 intercalated nucleic acid duplex. Similarly, one can use first and second enzyme labels where the product of one is a substrate for the other. The presence of complexes is then indicated by increased turnover by the second enzyme due to a proximate enzyme channeling effect. Other labeling schemes will be evident to one of ordinary skill in the art.
Alternatively, the antibody can be detected based on a native property such as its own antigenicity. A
labeled anti-~antibody) antibody will bind to the pri-mary antibody reagent where the label for the secondantibody is any conventional label as above. Further, antibody can be detected by complement fixa'Lion or the use of labeled protein A, as well as other techniques known in the art for detecting antibodies.

MS-13~0-CIP-~II

~;~3~

Where the antibody is labeled, as is preEerred, the labeling moiety and the antibody reagent are associated or linked to one another by direct chemical linkage such as involving covalent bonds, or by in-s direct linkage such as by incorporation o~ the label in a microcapsule or liposome which is in ~urn linked to the antibody. Labeling techniques are well-known in the art and any convenient method can be used in the present invention.

REA CTI OIV I~I XTIJRE

The test sample to be assayed can be any medium of interest, and will usually be a liquid sample of medical, veterinary, environmental, nutritional, or industrial significance. Human and animal specimens and body fluids particularly can be assayed by the present method, including urine, blood (serum or plasma), milk, cerebrospinal fluid, sputum, fecal matter, lung aspirates, throat swabs, genital swabs and exudates, rectal swabs, and nasopharnygal aspirates.
Where the test sample obtained from the patient or other source to be tested contains principally double stranded nucleic acids, such as contained in cells, the sample will be treated to denature the nucleic acids, and if necessary first to release nucleic acids f~om cells. Denaturation o nucleic acids is preferably accomplished by heating in boiling water or alkali treatment (e.g., 0.1 N sodium hydroxide), which if desired, can simultaneously be used to lyse cells. Also, release of nucleic acids can, for example, be obtained by mechanical disruption (freeze/
thaw, abrasion, sonication), physical/chemical dis-ruption ~detergents such as Triton, Tween, sodium MS-1320-CIP-~

~23~35~

dodecylsulfate, alkali treatmcnt, osmotic shock~ or heat), or enzymatic lysis (lysozyme, proteinase K, pepsin). The resultinx test medium will contain nucleic acids in single stran~e~ Form which can then be assayed according to the present hybridization method.
As is known in the art, valious hybridization conditions can be employed in the assay. Typically, hybridization will proceed at sligh-tly elevated temp-eratures, e.g., betwccn about 35 and 70C and usuallyaround 65C, in a solution comprising buffer at pH
between about 6 and 8 and with appropriate ionic ~ strength (e.g., 2XSSC where lXSSC = 0.15M sodium chloride and 0.015M sodium citrate, pll 7.0), protein such as bovine serum albumin, Ficoll (a trademark identifying a copolymer of sucrose and epichlorohydrin sold by Pharmacia Fine Chemicals, Piscataway, NJ), polyvinylpyrrolidone, and a denatured foreign DNA such as from calf thymus or salmon sperm. The degree of complementarity between the sample and probe strands required for hybridization to occur depends on the stringency of the conditions. Thc extent and speciEi-city of hybridization is affected by the following principal conditions:
1. The purity of the nucleic acid preparation.
2. Base compositlon oE the probe - G-C base pairs will exhibit greater thermal stability than A-T
base pairs. Thus, hybridizations involving higher G-C content will be stable at higher temperatures.

,~ .

~231~ 75 3. Length of llomologous base sequence - ~ny short sequence of bases (e.g., less than 6 bases), has a high degree of ~robability of being present in many nucleic acids. Thus, little or no specificity can be attained in hybridizations involving such short sequences. The present homolo~ous probe sequence will be at least l0 bases, usually 20 bases or more, and preferably greater than l0Q bases. From a prac~ical standpoint, the homologous probe sequence will often l0 be between 300-l000 nucleotides.
4. Ionic strength - The rate of reannealing in-creases as the ionic strength of the incubation solu-tion increases. Thermal stability of hybrids also in~reases.
5. Incubation temperature - Optimal reannealing occurs at a temperature about 25-30C below the melt-ing temperature (Tm) for a given duplex. Incubation at temperatures significantly below the optimum allows less related base sequences to hybridize.

6. Nucleic acid concentration and incubation time - Normally, to drive the reaction towards hy-bridization, one of the hybridizable sample nucleic acid or probe nucleic acid will be present in excess, usually l00 fold excess or greater.

7. Denaturing reagents - The presence of hy-drogen bond dîsrupting agents such as formamide and urea increases the stringency of hybridization.

8. Incubation time - The longer the incubation time the more complete will be the hybridization.

9. Volume exclusion agents - The presence of these agents, as exemplified by dextran and dextran sulfate~ are thought to effectively increase the con-centration of the hybridizing elements thereby increas-ing the rats of resulting hybridlzation.
MS- 132O CIP~
` ~;

~2~5i75 Normally, the antibo~y reagent, and the inter-calator in ~he case of formats wherein it is added as a ree compound, are not present in the hybri~ization solution, howcver7 this is not preclu~e~ where desired and where the hybridiz.ltion conditions are favorable to antibody binding and intercalation. In the usual case, intercalation complcxcs associa-ted with hybri-dized probe are ~etecte~ a~ter separa~ion of hybri-dized probe ~rom the hybri~ization solution. Wherc intercalator is added as a free compound, its concen-tration will normally be chosen so as to be sufficient to saturate the intercalation complexes present but not so great that signi~icant, e.g., greater than 10 percent, self-stacking of intercalator occurs. The conditions for intercalation will generally be mild, e.g., at a pH between about 6 and 8, moderate ionic strength ~<1), room temperature, ~ith no extended in-cubation necessary, i.e., less than 15 minutes in the usual case.
For detection of intercalation complex, anti-body to the complex is added in excess and al]owed to incubate for the time required to form a detectable product (e.g., 5 minutes to 24 hours) under condi-tions of neutral pH (e.g., between 6 and 8), moderate ionic strength ~-1) and moderate temperature (20-~0C).
Excess ~unbound) antibody :is then removed by washing under similar conditions.
It may be necessary or desirable to modify the procedure above by including an intercalator in the 30 washing step to maintain saturation oE the nucleic acid-intercalator complex. Also, if desired some or all of the steps above can be combined, such as add-ing the intercalating agent and antibody simultaneously.

MS-1320-CI`P-`II`

~L~23~5~5 R~'A (;E~T SYSTE~

The present invention additionally provides a reagent system, i.e., reagen~ combination or means, comprising all of the essential elements required to conduct a desired assay me~hod. The reagent system is presented in a commercially packaged form, as a composition or admixture where the compatability of the reagents will allow, in a test device configura-tion, or more usually as a test kit, i.e., a pack-aged combination of one or more containers, devices,or the like holding the necessary reagents, and usually including written instructions for the per-formance of assays. Reagent systems of the present invention include all configurations and compositions for performing the various hybridization formats described herein, particularly the four method types particularly illustrated above and in the drawings.
In all cases, the reagent system will comprise (1) a probe, (2) a nucleic acid intercalator as des-cribed herein, and (3) the antibody reagent, prefer-ably labeled with a detectable chemical group also as described herein. The system can additionally com-prise a solid support for immobilizing single stranded nucleic acids from the test medium. Alternatively, the probe element can be presented immobilized on such a support. ~urther, the intercalator can be present in the reagent system as a separate, free compound, substantially uncomplexed with nucleic acids, or can be bound to the probe either in the form of inter-calation complexes where the probe comprises a doublestranded region, and optionally covalently or other-wise chemically linked to one or both of the strands, or by being chemically linked, e.g., covalently, to a single stranded pr~be re~ion such that duplex forma-tion in suçh regicn results in the formation of ~23~35~5 intercalation complexes. In the case of the sandwich format, a second probe as described above is included in the system. A test kit form of the system can additionally include ancillary chemicals such as the components of the hybridization solution and denatura-tion agents capable of converting .louble stranded nucleic acids in a test sample into single stranded form. Preferably, there is included a chemical lysing and denaturing agent, e.g., alkali, for ~reating the sample to release single stranded nucleic acid there-from.
. 7 /

... .

~2~357S

The present invcntion will now be illustrated, bu~ is not intended to be limite~, by the following examples:
E~AMPLES

I. Ma t~r ia ~s A~ Preparation of tufA pro~e having a covalently intercalated double stranded portion.

The nucleic acid probe is a moclified M13mp9 vector (Messing and Vieria (1982~ Gene. 19:269; commercially available from New England Biolabs, Beverly, MA) con-taining an 800 bp insert bctween the Hinc II and EccRI
restriction endonuclease sites of RFMl3mp9. The 800 base insert is a fragment of the 1,190 base tufA
sequence from E. coli; and is the portion of the probe (which is comprised of vector and insert) which will actually hybridize to the specimen nucleic acid. It will be referred to as the tufA insert. The modified Ml3mp9 bacteriophage is denoted M13-10 and is available through the American Type Culture Collection, Rockville, MD (ATCC 39403-Bl).
The E. coli base organism used for propagation of Ml3-10 phage is JMl03 [~lac pro), supE, thi, strA, endA sbcB, hsdR , F'traD36, proAB, lacIq, ZQM15]
which is commercially available from Bethesda Research Laboratories, Gaithersburg, MD. E. coli JM103 is tr~nsformed with M13-10 DNA, and a culture of JM103 is subsequently infected with the transormed JM103.
The single stranded form of Ml3-10 is isolated from the phage particles excreted into the medium by the infected E. coli. The phage particles are harvested and tne single stranded M13-10 DNA is isolated follow-ing standard procedures ~Messing et al (1981) Nucleic Acids Res. 9:309].

' ~S-1320-CIP-II

.

.' ~3~35~S

Using an oligonucle~tide primer complementary ~o the M13mp9 vector on the 5' terminus of the tufA
insert, deoxynucleoside triphosphates and E. coli DNA polymerase (Klenow Fragment), ~ second DNA
strand i6 synthesized. This second strand is synthesized with limiting quantities of deoxynucleoside triphosphates such that it does not extend to the tufA insert because this insert must remain substantially single stranded for the probe to be useful in a hybridizat;on assay. This technique has been described in the literature [Hu and Messing (1982) Gene 17:271-277] and the oligonucleotide primer (sequence CACAATTCCACACAAC) is commercially available from New England Biolabs, Beverly, MA.
The amount of double stranded DNA present in the M13 10 probe can be estimated by using a radiolabeled nucleoside triphosphate in the second strand synthesis or by Sl nuclease digestion followed by a fluorescence assay with ethidium bromide.
The double stranded region of the M13-10 probe prepared as described above is intercalated and covalently linked with ethidium in a photoaffillity reaction using a photolabeled ethidium derivative, 8-azidoethidium. This photoreactive intercalator is prepared and isolated as described in the literature ~Graves et al (1977), Biochim. Biophys.
Acta 479:98-104]. Its binding to double stranded DNA has been shown to mimic that o~ its parent compound, ethidium bromide ~Bolton and Kearns tl978) Nucl. Acids Res, 5:4891; Garland et al (1980) Biochem. 19:3221 - our studies indicate that this procedure gives a mixture of 3-azido and 8-aæidoethidium isomers]. Because 8-azidoethidium ! ~ is photoreactive, standard precautions must be ~2~i7Si taken in handling it to prevent decomposition.
Working in the dark in the presence of a red photographic safelight has been found to be satisfactory. Solutions of 8-azidoethidium may be stored rozen in the dark at -70 C for at least one month.
Photolysis with visible light converts the azido moiety in 8-azidoethidium to a chemically reactive nitrene, which will quickly react with available nucleophiles to form covalent ethidium adducts [Knowles (1971) Acc. Chem. Res. 5:155]. If 8-azidoethidium is intercalated between the base pairs of DNA when photolysis occurs, covalently coupling of ethidium to DNA occurs with high efficiency [Bolton and Kearns (1978) Nucl. Acids Res. 5:48911.
Ethidium is covalently coupled to the double stranded region of M13-10 by photolysis of a solution containing approximately lmM DNA base pairs and 0.5mM 8-azidoethidium in an appropriate buffer such as 20 mM
tris-(hydroxymethyl)aminomethane (Tris-HC1), 200 mM
sodium chloride (NaCl), pH 8Ø Photolysis is accomplished with a 150 watt outdoor spotlight, with the stirred reaction 5-20 centimeters (cm) away from the light source. To prevent the photolysis reaction from overheating and to block out any short wavelength radiation, i.e., less than 300 nanometers (nm), the photolysis reaction is surrounded by a glass water bath which is connected to a water circulator with temperature regulation After an appropriate incubation period such as 60 minutes, ethidium groups not covalently bound to DNA are removed by a series, e.g., 10, of MS-1320-CIP~

s~s successive extractions with an equal volume of water saturated n-butanol. ~dditional 8-azidoethidium (inal concentration in the ran~e of 0.4 mM) is added and the photolysis and extraction steps are repeated.
The amount of ethidium associated with the DNA is es-timated using extinction coefficient values o~
~490- ~ x 103 M 1 cm 1 for photolyzed ethidium a2ide the relationship between A260 and A4go for photolyzed ethidium bound to DNA [A 60 = (A490 x 3.4)-0.011], and E260- 1.32 x 104 M ~ cm 1 for the concentration of DNA base pairs of a given DNA being labeled. Prefer-ably, the probe is saturated with ethidium such that there is 1 ethidium moiety for every 2 DNA base pairs in the double stranded region of the probe. The photolysis reaction and extractions are repeated until the desired labeling density is obtained.

B. Preparation of adenovirus probes for sandwich hybridization ~ormat.

Sandwich hybridization formats are described in the literature - Dunn and Hassell (1977) Cell 12:23;
Dunn and Sambrook (1980) Methods in Enzymology 65:468, Ranki et al (1983) Gene 21:77; Ran~i et al (1983) Curr. Topics in Microbiology and Immunol. 104:307-310.
This approach requires two nucleic acid probes, each of which is complementary to a unique region o~
the nucleic acid being kested in a specimen. One o~ the probes is immobilized on a solid support while the other is labeled in some manner and is initially in solution with the specimen nucleic acids. These will be referred ko as the solid and solution phase probes, respectively.

MS-1320-CI~

The solid and solution phase probes are prepared from restriction endonuclease digests of DNA from adenovirus type 2 (Ad2) as described by Ranki et al (1980) Gene 21:77-85. The solid phase probe is comprised of BamHl fragments C or D [Tooze (1980) "The Molecular Biology of Tumor Viruses"
(2nd ed) Part 2: DNA Tumor viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp.
933-9343 of Ad2 DNA inserted in a pBR322 vector.
These probes have been denoted pkTH1201 and pkTH1202, respectively. The solution phase probe is comprised of a BamHl and BglII restriction endonucleoase digest of pkTH1201 in which fragments are shotgun-cloned into the BamHl restriction endonuclease site of M13mp7 [Messing et al (1981) Nuc. Acids Res. 9:309]. The modified M13mp7 containing as an insert a fragment of the Ad2 C
fragment is designated mkTH2306.
The solution probe mkTH1206 is made partially double stranded as described in Part I-A above and the double stranded region is labeled with an intercalating agent, e.g., ethidium, also as described in Part I-A above.

II
MS-1320-CIP ~

C. Preparation of HCMV probe.

The EcoRI restriction endonuclease fragment O
from human cytomegalovirus (HCMV) strain AD169 [Tamashiro et al (1982) J. Virol., May, 547-556;
Chou and Merigan (1983) New Engl. J. of Med. 308:921]
is cloned into the pBR322 derivative pACYC184 which is used to transfect E. coli strain HB101 Rec A as described by Tamashiro et alO After propagation and purification at the insert-bearing pACYC184, the plasmid is digested with restriction endo-nuclease EcoRI and the 6.8 kb O fragment of HCMV
is purified by preparative electrophoresis in 0.8%
agarose gels using standard procedures [Maniatis et al (1982) "Molecular Cloning", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY].

D. Preparation of intercalator-labeled HCMV probe.

The purified double-stranded O fragment from Part I-C above is then covalently labeled with ethidium by using the photolabile ethidium derivative, 8-azidoethidium, as described in Part I-A above.

~ .

~23~

E. Preparation of intercalation complex immunogen.

Calf thymus or salmon sperm DNA is sheared by repetitive passage through a hypodermic needle, treated with Sl nuclease to remove single stranded regions [Maniatis (1982) "Molecular Cloning'l, Cold Spring Harbor Laboratoryl Cold Spring Harbor, NY] and separated from the resulting nucleotides by any one of a number of st~ndard methods (e.g., ethanol precipitation, gel exclusion chromatography, or ion exchange chromato-graphy).
The purified double stranded DNA is then covalentlycoupled to the photolabile ethidium derivative ~-azidoethidium by photolysis as described in Part I-A
above. A carrier protein is prepared by methylation of carboxylic acid residues (Mandell and Hershey (1960) Anal. Biochem. 1:66) then combined with the intercator-labeled DNA to form an electrostatically associated nucleic acid-protein complex as described in Poirier et al (1982) PNAS 79:6443.

F. Preparation of polyclonal antiserum to the intercalation complex.

Polyclonal antiserum against intercalator DNA
complexes is elicited in rabbits using the immuniza-tion techniques and schedules described in the litera-ture ~Stollar (1980) Methods in Enzymology 70:70].
The antiserum is screened in a solid phase assay similar to that used for monoclonal antibodiesl e.y., as des-cribed by Lange et al (1976) Clin. Exp. Immunol. 25:
191; Pisetsky et al (198~1) J. Immun. Methods 41:187.
The initial screening criterion would be binding to the intercalator-DNA complex.

. .
'' ~3~

The IgG fraction of the antisera containing anti-bodies is isolated from other serum proteins by ammonium sulfate precipitation followed by chromato-graphy on DEAE cellulose [Livingston (1974) Methods in Enzymology 34:723].
The IgG fraction of the antisera is purified further by affinity chromotography on a column con-taining a resin on which the DNA-intercalator complex is im~obilized [Stollar (1980) Methods in Enzymology 70:70]. After applying the IgG fraction to the column, nonspecifically bound protein is removed by washing, and the specific antibodies eluted with 2M
acetic acid in the cold [Stollar (1980) ibid].
The purified antibodies are screened more thor-oughly to determine their usefulness in the hybridiza-tion assay. The antibodies must bind the intercalator-DNA complex with high affinity (preferably, KA> 10 M ); cross-reactivity with free intercalator or single stranded DNA is not acceptable. Depending upon the assay format, some cross-reactivity of the antibodies with double stranded DNA is acceptable.

G. Preparation of monoclonal antibodies to the intercalation complex.

Using the intercalator-DNA immunogen prepared as described above, mouse monoclonal antibodies to the intercalator-DNA complex are obtained using stan~ard procedures [GalEre and Milstein (1981) Methods in Enzym. 73:1]. The monoclonal antibodies are screened using a modification of the techniques described in the literature, e.g., Lange et al (1976) Clin. Exp.
Immunol. 25:191; Pisctsky et al (1981) J. Immun.
Methods 41:187). To be useful in the assay for detec-tion of DNA-intercalator complexes, a monoclonal anti-body should bind to the DNA-intercalator complex , ~.

3~ S

with high affinity (preferably, KA> 11 M 1), but cannot bind to single stranded DN~ or free inter-calating agent. Cross-reactivity with dcuble stranded DNA may be acceptable in some of the assay formats.
Mouse monoclonal antibody is purified in a two step procedure. The neat ascites fluid is applied to a column of Affi-Gel Blue resin (Bio-Rad Laboratories, Richmond, CA) equilibrated with lOmM Tris-HCl, 0.15M
NaCl, pH 8.0, and eluted with the same buffer. This step removes albumin, which is retained on the column.
The final step in the purification is application to DEAE-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) and elution with a linear gradient of lOmM Tris-HCl, PH 8.0, to 10 mM Tris-ElCl, 200mM NaCl. This gives purified mouse monoclonal antibody free from contaminat-ing serum proteins such as albumin and transferrin.

H. Preparation of ~-galactosidase-antibody conjugate.

3-galactosidase (30,000 units, grade VIII, commercially available from Sigma Chemical Co., St.
Louis, MO) was dissolved in 2ml of a buffer solution comprised oE O.lM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), O.O9M NaCl, pH 7Ø
This gave a ~-galactosidase solution containing 37.7 mg o~ protein (70 nmol) in 1~8~ ml. ~ 3.5 ~mol portion (a 50-fold molar excess) oE dithiothreitol (DTT) was added to this solution, and the mix-ture allowed to stand at room temperature for four hours.
DTT was removed from the enzyme solution by chromatographing the mixture on a 2.5 x 80 cm column of Sepharose 6B Cl resin (Pharmacia Fine Chemicals, Piscataway, NJ) using as the eluent the HEPES/NaCl buffer described above. Protein-containing fractions were pooled to give a total volume of 15 ml. Using an :, .

.

23~

El80 = 20.9 ~Worthington Enzyme ~lanual (1977), Worthington Biochemical Corporation, Freehold, N~, p. 195], the ~-galactosidase concentration was deter-mined to be 9.62 mg/ml. Th~ number of sulfhydryl groups on ~he enz~ne was determined to be ll.0 using Ellman's reagent [Ellman ~1959) Arch. Biochem.
Biophys. 82:70]. Typicallv this protocol gives 9-15 free sulfhydryl groups per ~-galactosidase molecule.
The heterobifunctional coupling reagent

10 succinimidyl-4-~N-maleimidomethyl)cyclo]lexane-l-carboxylate ~SMCC, available from Pierce Chemical Co., Rockford, IL) was used to collple ~-galactosidase to an antibody. This coupling reagent contains a maleimido group which selectively reacts with sulfhy-dryl moieties and an N-hydroxysuccinimide ester for coupling to amino groups. The coupling procedure is comprised of two steps: reacting SMCC with antibody amino groups followed by coupling the derivatized antibody to ~-galactosidase by reaction of the malei-mido moiety with ~-galactosidase sulfhydryl groups.
A 5.3 mg portion of SMCC was dissolved in 250 ~l of anhydrous N,N-dimethylformamide ~DMF). The actual concentration of reactive maleimide groups in this solution was determincd by reaction with a known quantity of glutathione, followed by determining the quantity of glutathione sulfhydryl groups using Ellman's reagent ~ibid). For example, ~0 ~l of the DMP solution was diluted to 3 ml with }lEPES/O.Ol5 M
NaCl buffer. A 25 ~I volume of this aqueous solution of SMCC solution was then combined with 825 ~l HEPEStNaCl buffer and lO0 ~l of lmM glutathione.
After standing at room temperature for l5 minutes, the amount of unreacted glutathione was determined using Ellman's reagent ~ibid) and the appropriate standards (i.e., unreacted glutathione and a blank with no ~3857S

glutathione). Sevcral determinations were made for each SMCC solution, and their results averaged. This protocol indicated that the ~MF solution of SMCC pre-pared as describe~ above was 52mM in reactive maleimide groups.
A 6.0 mg ~40 ymol) portion of a mouse~monoclonal antibody was combined with 400 ~mol of SMCC in a final ~olume of 533 ~1 of HEPES/0.15M NaCl and allowed to react 1 hour at 30C. The reaction mixture was then applied to a 1 x 24 cm column of Bio-Gel P-2 resin (Bio-Rad Laboratories, Richmond, CA) and eluted with HEPES/0.15M NaCl. All protein containing fractions were pooled; the protein concentration was determined using the method of Sedmack and Grossberg ~Anal.
Biochem. 79:544(1977)] and the number of maleimide groups was determisled as described above. These determinations indicated an antibody concentration of 1.98 mg/ml, with 1-2 maleimides/antibody molecule.
A 28 mg portion of the antibody-maleimide conju-gate was combined with 10 mg of DTT-treated ~-galactosidase (final ~olume 2.45 ~1) and allowed to react 4 hours at room temperature. The mixture was then applied to a 2.5 x 80 cm columsl of Sepharose 6B Cl (Pharmacia, Piscataway, NJ) and eluted with HEPES/0.15M NaCl at 4C. The flow rate was 4 ml/hr;
3 ml fractions were collected. Fractions were assayed ~or ~-galactosidase activity and antibody binding capacity. Fractions 39-42 had both properties and were pooled.

* Trade Mark ;7~

J. Preparation of biotin-labeled antibodies.

Purified antisera is treated with the N-hydroxysuccinimide ester of biotin ~commercially available from Sigma Chemical Co., St. Louis, MO or Biosearch, San Rafael, CA) using the methods described in the literature ~Oi et al (1982), J. Cell~ Biol.
93:981; Heitzmann et al ~1974) Proc. Natl. Acad. Sci.
~SA 71:3537; Green (1975) A~v. Protein Chem. 29:85].

K. Preparation of radiolabelled antibodies.

Purified antibody is radiolabeled following pro-cedures given in the literature. Radioiodination is accomplished by reaction of the antibodies with 125I-labeled 3-~4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester ~commercially available from New England Nuclear, Boston, MA) following the protocol of Bolton and Hunter [Biochem. J. 133:529 (1973)]. Alternatively, the antibody fraction is covalently coupled with a bifunctional chelating agent ~Yeh et al ~1979) Anal. Biochem. 100:152] and is sub-sequently labeled with an appropriate radioactivemetal ion. This latter approach has the advantage that the shelf life of the antibody fraction is not limited by the half life of a radioisotope.

MS-1320-CIp-II

~ ~3~S7S

L. Preparation of alkaline phos~hatase-biotin-avidin complex.

An alkaline phosphatase-biotin-avidin complex is prepared as described by Leary et al [Proc. Na~l. Acad.
Sci. USA 80:4045 ~1983~] Calf intestinal alkaline phos-phatase is first cross-linked by reaction with di-succimidyl suberate, then coupled with the N-hydroxysuccinimide ester of biotinyl-~-aminocaproic acid. After purification, the alkaline phosphatase-biotin complex is labeled with avidin ~which has 4 biotin binding sites/avidin molecule) by combining the alka-line phosphatase-biotin complex with a slight molar excess of avidin. Either avidin or a bacterial analog of avidin, steptavidin ~Hofmann et al (1980) Proc. Natl.
Acad. Sci. USA 77:4666-4668; commercially available from Bethesda Research Laboratories, Gaithersburg, MD]
may be used in this last step.
The detection system used for the alkaline phosphatase-biotin-avidin complex is comprised of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as described by Leary et al (ibid).

MS-1320~CIp-II

, .

:~l2~3~57~

Il. Methods A. Detection of gram negative bacteria in urine -~Method Type 1) solid-phase, sample immobilized, hybridization assay with tufA probe having a covalently intercalated double stranded region, monitored with enzyme-labeled antibody (see Fig. 2).

Because its sequence is highly conser~ed, the tufA
sequence from E. coli can be used to detect the presence of gram negative bacteria in urine specimens.
Clinical urine samples are clarified by centrifuga-tion for a short period of time ~e.g., 5 min.) at a low centrifugal force (e.g., 8000 rpms with a Sorvall GLC-3 centrifuge). Bacterial cells in the supernatant are lysed and the bacterial genome is denat~lred by making the urine specimen 0.5 M in sodi~m hydroxide (NaOH) for 10 minutes at an elevated temperature (65C). Alter-natively, this may be done by heating the urine to 90C and maintaining that temperature for 10 minutes.
After lysis and denaturation, the urine sample is diluted and neutralized with an equal volume of 20XS~PE (3.6M NaCl, 0.2M NaPO~, 20mM EDTA, pH 7.7).
The urine specimen is then immediately filtered through a nitrocellulose membrane under mild vacuum. The immobilized bacterial DNA is then fixed to the nitro-cellolose membrane by baking in vacuo at 80C ~or 2 hours. The filter containing the immobilized speci-men DNA is treated with prehybridization solution ~0.1~
(w/v) each Ficoll ~Pharmacia), polyvinylpyrrolidone and ~ BSA in SXSSPE, 100-200 ~g/ml denatured7 haterologous DNA] for 1-3 hours at 65C. A 50-100 ~1 vol~me o~ pre-hybridization solution/cm2 of filter is used~ After prehybridization treatment, the ethidium labeled probe prepared as described in Part I-A above is added to the prehybridization solution and hybridization is ~S-132~-CII'-~I

~3~3~7S

allowed to occur (1-72 hours). The above are all standard techniques found in the literature [Maniatis et al (1982) "Molecular Cloning", Cold Spring Harbor Laboratory, Cold Spring ~larbor, NY].
After the hybridization, the filter is washed to remove excess probe DNA. The fil~er is then immersed into a solution containing ~-galactosidase-labeled anti-bodies to ~he intercalator-DNA complex and incubated for 5 minutes to 12 hours. Excess antibody is removed by washing, and the amount of ~-galactosidase associated with the filter is determined by adding a fluorogenic substrate of the enzymes (e.g., ~-methylumbelli~erone ~-galactoside) and measuring fluorescence intensity after a period of time. Be-cause the quantity of enzyme present is likely to bequite low, the fluorogenic substrate is added in a concentration greater than or equal to its Michaelis constant (Km) for ~-galactosidase. Standards, with a defined quantity of probe immobilized on the ~ilter, can be run simultaneously so that hybridization can be quantitated.

B. Detection of adenovirus - (Method Type 2) sandwich hybridization assay with labeled probe having a covalently intercalated double stranded region, monitored with enzyme-labeled antibody (cee Fig. 3)-This method is based on the sandwich hybridization assay described by Ranki et al for the detection of adenovirus type 2 (Ad2) DNA in clinical samples 30 ~Ranki et al (1983) Gene 21:77; RanXi et al ~1983) Current Topics in Microbiology and Immunology 104, Springer-Verlag, NY p. 307]. The solid phase probe pKTH1202 (see Part I-B above) is denatured, nicked and immobilized on nitrocellulose filters. A~ter , .

~ 2~ ~
fixation (baking in V-ICUO at 80C -for 2 hours), the filters are treated with a prehybridization solution for one hour at 65C. DNA from clinical specimens and the intercalator-labeled solution hybridization probe mkTH1206 ~prepared as described in Part I-B above~ are added to the prehybridization solution and hybridiza-tion of the probes with the specimen DNA is allowed to occur for 1-72 hours. After hybridization, excess solution probe (mkTH1206) is removed by washing.
0 The extent of hybridization is quantitated using ~-galactosidase-labele~ antibody to the intercalator-DNA complex as outlined in Part II-A above.

C. Detection of human cytomegalovirus in urine (Method Type 3) solid-phase, probe immobilized hybridization assay, monitored with biotin labeled antibodies and enzyme-labeled avidin (see Fig. 4).

This method is used for the detection of human cytomegalovirus (HCMV) in clinical urine specimens.
The purified probe (EcoRl O fragment of HCMV strain AD169, as described in Part I-C above) is denatured by heating at 90C for 10 minutes, rapidly chilled on ice (to prevent renaturation) and combined with an equal volume of 20XSSPE (3.6M NaCl, 0.2M NaPO4, 20 mM EDTA, pH 7.7). The single stranded probe DNA, is then immobilized and fixed on a nitrocellulose membrane using standard procedures. The membrane is then treated with a prehybridization solution, prefer-ably one not containing het,erologous DNA. One pre-hyb-ridization solution which C~ll be used is that des-cribed by N,ew England Nuclear for their Cene Screen PlusTM membranes; this solution is comprised of 1~
SDS, lM NaCl, and 10% dextran sulfate. To prevent nonspecific binding of the antibody in the final steps of the detection schemes, it may be desirable to include BSA in the prehybridization solution.
MS-1320-CIP~

~3B5~S

The clinical urine specimen to be tested is prepared in a manner similar to that described by Chou and Merigan ~New ngl. J. Med. 308:921 ~1983)].
After clarification of the sample and concentration of the HCMV phage particles by centrifugation, they are resuspended in a minimum vol~lme of 0.5M NaO~I and allowed to stand for 15 minutes. After neutraliza-tion with a minimwn volume of 20XSSP~, the denatured clinical specimens are adde~ to the filter in 1% SDS~
lM NaCl, 10% dextran sulfate and 100 ~g/ml denatured salmon sperm DNA. Ilybridization is allowed to proceed at 65C for 1-72 hours; the filters are then washed in 2XSSPE.
The filters are immersed in a minimum volume of a solution containing the selected intercalator (e.g., ethidium bromide at a submillimolar concentration).
Biotinylated antibody to the DNA-intercalator complex (Part I-J above) is then added and allowed to bind (1-24 hours). Excess antibody is removed by washing.
In some situations it may be necessary to include ~he intercalating agent in these wash steps to keep the double-stranded DNA saturated.
A streptavidin-biotin-alkaline phosphatase com-plex (Part I-L above) is ~hen added and allowed to bind to the biotinylated antibody associated with the DNA as described by Ward et al [Proc. Natl. Acad. Sci.
USA 80:4045~1,983)]. After washing away excess alkaline phosphatase conjugates, the presence of conjugate associated with the filter is determined by ~dding a colorimetric substrate Eor alkaline phos-phatase as described by Ward (ibid). This is a direct measure of the presence o~ ~ICMV DNA in the clinical urine specimen.

MS-1320-CIP~
~, .
., ~8~7S

D. Detection of human cvtomegalovirus in urine (Method Type 4) solid-phase, intercalator-labeled-probe immobilized hybridiza~ion assay, monitored with radiolabeled second antibody to intercalation complex antibody (see Fig. 5).

This method is similar to that of Par~ II-C above except that the probe is already lab~led with inter-calating agent, and the final step of the detection scheme requires a second, isotopically labeled antibody.
The probe, ethidium labeled Eco RI fragment O o~-HCMV (prepared as describcd in Part l-D above) is denatured, immobilized and fixed on a nitrocellulose support as described for the method oE Part II-C above.
Viral DNA is isolated from urine samples, denatured, and hybridized to the immobilized probe also as described in Part II-C above, except that addition of free intercalator is unnecessary.
After ~ashing the filter with the hybridized DNA, excess mouse monoclonal antibody to the intercalator-DNA complex (see Part I-G above) is added and allowed to bind to the hybridized DNA intercalator complex (30 minutes to 6 hours). Excess mouse antibody is re-moved by washing and excess radiolabeled rabbit-anti(mouse IgG) (Part l-K) is added. After a 30 minute to 6 hour incubation, excess antibody is again removed by washing. Hybridization is quantititated by auto-radiography or gamma counting.

MS-1320 CIp-II

38~

III. Demonstration o~ Antigenicit~ of IntercaZation CompZe~es A. Preparation of covalent ethidium-DNA complexes A~out 250 mg of salmon sperm DNA (Sigma Chemical Co., St. Louis, MO) is dissolved in 40 ml of 50 mM
NaCl and sheared by five passages through a 23 gauge needle. The sheared DNA is placed in a 250 ml flask and diluted with an additional 160 ml of buffer. One hundred forty-five microliters (145 ~1) of Sl-nuclease, 200,000 units per ml (Pharmacia P-L Biochemicals, Piscataway, NJ), is added and the mixture is incubated at 37C for 50 minute 3 .
Then the reaction mixture is extracted twice with phenol:chloroform, once with chloroform and the DNA is precipitated twice with ethanol ~Maniatis et al (1982) "Molecular Cloning", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY]. The final precipitate is dissolved in 70 ml of 20 mM Tris hydrochloride buffer, pH 8Ø
2Q This DNA is reacted with 8-azidoethidium under the following conditions. The reaction mixture is prepared with 33 ml of 2.7 mg DNA/ml, 13.5 ml of 4.95 mM 8-azidoethidium, 13.5 ml of 0.2 M
Tris-hydrochloride buffer, pH 8.0,0.2M NaCl, and 76 ml water. The mixture is placed in a 250 ml beaker with a water jacket m2intained at 22C. The mixture is stirred and illuminated for 60 minutes by a 150 watt spotlight at a distance of 10 cm. This photolysis is repeated with an identical reaction mixture.

Ms-l32o-cI`
' ' The photolyzed reaction mixtures are combined and extracted 10-times with an equal volume each time o~ n-butanol saturated with 20 mM Tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl. The ex-5 tracted DNA solution is combined with 23 ml of 4.95 mM 8-azidoethidium and 77 ml of 20 mM
Tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl. This solution is stirred in the water-jacketed beaker and photolyzed for 90 minutes. The reaction products are extracted 10 times with buffer saturated butanol as described above and the DNA is precipitated with ethanol. The precipitate is dissolved in 10 mM
Tris~hydrochloride buffer, pH 8.0, 1 mM EDTA and the absorbances at 260 and 490 nm are recorded. Calcula-tions made as described in Example lA above indicate1 ethidium residue is incorporated per 4.5 DNA base pairs.

B. Preparation of methylated thyroglobulin One hundred milligrams of bovine thyroglobulin (Sigma Chemical Co., St. Louis MO) is combined with 10 ml of anhydrous methanol and 400 ~1 of 2.55 M HCl in methanol. This mixture is stirred on a rotary mixer at room temperature for 5 days. The precipitate is collected by centrifugation and washed twice with methanol and twice with ethanol. Then it is dried under vacuum overnight. About 82 mg of dry powder is obtained.

Ms-l32o-cI~II

3~2~5~

.

C. Preparation of covalent ethidium DNA methylated thyroglobulin complex Fifty milligrams (55 mg) of methylated thyro-globulin is dissolved in 10 ml of water and 11.3 ml of a 2.2 mg/ml covalent ethidium DNA solution is added.
A precipitate forms immediately and the suspension is diluted with 5.0 ml of 1.5 M NaCl and 24.6 ml water.

D. Immunization of rabbits Two milliliters (2 ml) of a mixture composed of 2.5 ml of the covalent ethidium-DNA methylated thyroglobulin complex, 2.5 ml of 0.15 M saline and 5.0 ml of complete Freunds adjuvant is injected into Eour subcutaneous sites on a New Zealand white rabbit. Three weeks later a similar immunization with incomplete Freunds adjuvant is administered followed by additional immunizations at 4 week intervals. Fourteen weeks after the initial immunization, blood is collected for preparation of antiserum.

MS-1320-CI~I~

5'~S

~ 58 -E Titratlon of antibody to ethidium-DNA

Antiserum to covalent ethidium-DNA is titered by an enzyme label immunosorbant assay. Polynucleo-tides are adsorbed onto the walls of polystyrene microtiter plates and then the rabbit antibody is allowed to bind. Finally the antibody is detected with peroxidase labeled goat anti-rabbit IgG.
Fifty microliter (50 ~1) aliquots of solutions containins 5 ~g o~ polynucleotide per ml in 15 mM
sodium citrate, pH 7.0, 0.15 M NaCl is dispensed into wells of Immulon II microtiter plates (Dynatek, Alexandria, VA) and shaken gently at room temperature for 2 hours. Then the wells are emptied and washed with 10 mM sodium phosphate bu~er, pH 7.4, 0.15 M NaCl, 0.5~ bovine serum albumin and 0.5% Tween 20 (PBS/BSA/Tween).
Rabbit antiserum is diluted into 10 mM sodium phosphate, pH 7.~, 0.15 M NaCl, 0.5% BSA and 50 ~1 aliquots are added to the wells and allowed to stand for 30 minutes. The wells are washed three times with PBS/BSA/Tween. Peroxidase covalently coupled to goat-antirabbit IgG (Cappel Laboratories, Cochranville, PA) is diluted 500-~old in 10 mM sodium phosphate, pH 7.4, 0.15 M NaCl, 0.5% BSA and 50 ~1 ali~uots are added to each well. This solution is allowed to stand in the wells for 30 minutes at room temperature and then the wells are washed three times with PBS/BSA/
rrween .

MS-1320-CIP~II

, ~.23l~57~;

One hundred mlcromolar (100 ~M) ethidium bromide i5 included in the diluted antiserum of wells contain-ing noncovalent ethidium-DNA complex and the ethidium control wells. All wash solutions and reagents des-cribed above for processing these wells contain 100 ~M ethidium.
A peroxidase substrate solution is prepared with:
20 mg o-phenylenediamine 5 ml 0.5 M NaHPO4 12 ml 0.1 M sodium citrate 13 ml water 20 ~1 30% hydrogen peroxide Seventy-five microliters (75 ~1) of substrate solution is added per well and allowed to react for 10 minutes at room temperature. The reactions are quenched by addition of 50 ~1 of 2.5 M sulfuric acid. Then the absorbances at 488 nm are recorded with a Artek Model 210 microliter plate photometer 2Q (Dynatek, Alexandria, VA).
Normal rabbit serum is used as a control and is processed as described for the rabbit antiserum.

* Trade Mark MS-1320-CIP-lI

;: ~

~;~3~S7S

F. Results The results are given in Table ~ and show that antibody in the control rabbit serum does not bind at significant levels to any of the coated or uncoated wells. It might have a weak antibody titer to single stranded DNA.
The antiserum to the covalent ethidium-DNA has very high titer to the covalent ethidium-DNA. Part of these antibodies are probably binding to ethidium residues that are coupled covalently to the phosphate ribose chain. This conclusion is based on the obser-vation that the titers to the noncovalent ethidium-DNA complex are much lower (see Table A).
These results demonstrate that antibodies can be raised to the ethidium-DNA intercalation complex which do not crossreact significantly with native single or double stranded nucleic acid.

MS-1320-cIP-~rI

' ~ ' ' ..-,~

~3~ 5 T~ble A
Dilution Absorba~ccs (488 nm) buffer Cov~lent Doublc-strand Noncovalent Ethidi~lm Single-strand Antiserum Control ~thidium-DNA DNA Ethidium-DNA Control DNA
S0 0.067 ~1.2 0.126 0.825 0.0~9 0.283 200 0.032 ~1.2 0.06B 0.597 0.021 0.~84 800 0.022 ~1.2 0.067 0.30 O.O~fi 0.~74 Control Serum S0 0.038 0.053 0.091 0.031 0.023 0.245 0 200 0.025 0.044 0.032 0.016 0.017 0.181 800 0.017 0.034 0.054 O.OlS 0.016 0. 190 Notes:
1) The buffar control does not contain DNA on the wells, 2) Double-stranded DNA contains cale thymus DNA on the wells, 3) Noncovalent ethidium-DNA has calf thymus double-stranded DNA on the wells and 100 ~IM ethidium in the reagent and wash solutions.
4) Ethidium control does not have DNA on the wells hut has 100 ~M ethidium in the reagent and wash solutions.
S) The single-stranded DNA has heat denatured calf thymus DNA coated on the wells.

The present invention has been particularly described and exemplified above. Obviously, many other variations and modifications of the invention may be made without departing from the spirit and scope hereof.

.

Claims (43)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for detecting a particular poly-nucleotide sequence in a test medium containing single stranded nucleic acids, comprising the steps of:
(a) combining the test medium with (i) a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to hybridization between the sequence to be detected and the complementary sequence in the probe, and (ii) a nucleic acid intercalator capable of binding to double stranded nucleic acid in the form of inter-calation complexes, and (b) detecting hybridized probe by adding an antibody, or a fragment thereof, capable of binding with intercalation complexes in the hybridization product resulting from step(a), and determining the antibody or fragment thereof which becomes bound to such complexes. , -

2. The method of Claim 1 wherein the inter-calator is combined with the test medium as a separate, free compound and noncovalently binds with double stranded nucleic acid to form intercalation complexes.

3. The method of Claim 1 wherein the intercala-tor is chemically linked to the probe in the single stranded complementary region of the probe, whereby upon hybridization said intercalation complexes are formed in such region.

4. The method of Claim 1 wherein the antibody or fragment thereof is labeled with a detectable chemical group.

5. The method of Claim 4 wherein the detectable chemical group is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

6. The method of Claim 1 according to a solid phase hybridization technique wherein one of the probe and the single stranded nucleic acids from the test medium is immobilized on a solid support and wherein the antibody associated with the solid support is determined.

7. The method of Claim 1 according to a solid phase sandwich hybridization technique wherein the test medium is combined with first and second nucleic acid probes each comprising at least one single stranded base sequence which is substantially complementary to a mutually exclusive portion of the sequence to be detected and wherein one of the probes is immobilized on a solid support or comprises a binding site for a binding substance and is thereafter rendered immobilized by the presence of an immobilized form of such binding substance.

8. The method of Claim 1 according to a solution phase hybridization technique wherein the probe comprises a binding site for a binding substance and wherein after the hybridization step there is added an immobilized form of such binding substance.

9. The method of Claim 8 wherein the probe comprises a biotin or hapten moiety and the binding substance is avidin or an anti-hapten antibody, respectively.

10. The method of Claim 1 wherein the probe additionally comprises a double stranded portion which, upon addition of the intercalator in step(a) as a separate, free compound, forms said intercalation complexes.

11. The method of Claim 1 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.

12. A solid-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:
(a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, one of the probe and the single stranded nucleic acids from the test medium being immobilized on a solid support, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence, (b) contacting the solid support carrying resulting immobilized duplexes with a nucleic acid intercalator and an antibody, or fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, (c) separating the solid support carrying resulting immobilized antibody or fragment thereof from the remainder of the reaction mixture, and (d) determining the separated antibody or fragment thereof on the solid support as an indication of the presence of the sequence to be detected in the liquid test medium.

13. The method of Claim 12 wherein prior to step (b) the solid support carrying immobilized duplexes resulting from step(a) is separated from the remainder of the reaction mixture.

14. The method of Claim 12 wherein the antibody or fragment thereof is labeled with a detectable chemical group and wherein in step(d) such detectable group is measured on the solid support as an indication of the presence of the sequence to be detected.

15. The method of Claim 12 wherein the probe also comprises at least one double stranded region which, upon addition of the intercalator in step(b), forms said intercalation complexes capable of being hound by the antibody or fragment thereof.

16. The method of Claim 12 wherein the liquid test medium comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.

17. A solid-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:
(a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe, the probe comprising at least one single stranded base sequence substantially complementary to the sequence to be detected and the probe being chemically linked to a nucleic acid intercalator in the single stranded complementary region of the probe such that duplex formation in such region bearing the linked intercalator results in the formation of intercalation complexes, one of the probe and the single stranded nucleic acids from the test medium being immobilized on a solid support, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence, (b) adding to the reaction mixture an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, (c) separating from the remainder of the reaction mixture, the solid support carrying resulting immobilized antibody or fragment thereof bound to intercalation complexes formed between the intercalator-linked probe and the sequence to be detected, and (d) determining the separated antibody or fragment thereof on the solid support as an indication of the presence of the sequence to be detected in the liquid test medium.

MS-1320-CIP-II

20. A solution-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:
(a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, the probe comprising a binding site for a binding substance, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence, (b) adding to the reaction mixture simultaneously or in separate steps (i) a nucleic acid intercalator, (ii) an antibody, or fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and (iii) an immobilized form of a binding substance for the probe, (c) separating the resulting immobilized phase comprising antibody, or fragment thereof, bound to immobilized intercalation complexes from the remainder of the reaction mixture, and (d) determining the separated immobilized antibody, or fragment thereof, as an indication of the presence of the sequence to be detected in the liquid test medium.

MS-1320-CIP-II

20. A solution-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:
(a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, the probe comprising a binding site for a binding substance, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence, (b) adding to the reaction mixture simultaneously or in separate steps (i) a nucleic acid intercalator, (ii) an antibody, or fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and (iii) an immobilized form of a binding substance for the probe, (c) separating the resulting immobilized phase comprising antibody, or fragment thereof, bound to immobilized intercalation complexes from the remainder of the reaction mixture, and (d) determining the separated immobilized antibody, or fragment thereof, as an indication of the presence of the sequence to be detected in the liquid test medium.

21. The method of Claim 20 wherein the antibody or fragment thereof is labeled with a detectable chemical group and wherein in step(d) such detectable group is measured in the immobilized phase as an indication of the presence of the sequence to be detected.

22. The method of Claim 20 wherein the probe comprises a biotin or hapten moiety and the immobilized binding substance is avidin or an anti-hapten antibody, respectively.

23. The method of Claim 20 wherein the liquid test medium comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.

24. A reagent system for detecting a particular polynucleotide sequence in a test medium, comprising:
(1) a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, (2) a nucleic acid intercalator, and (3) an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator.

25. The reagent system of Claim 24 wherein the antibody or fragment thereof is labeled with a detectable chemical group.

26. The reagent system of Claim 25 wherein the detectable chemical group is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

27. The reagent system of Claim 25 wherein the detectable chemical group is an enzyme.

28. The reagent system of Claim 24 which additionally comprises a solid support for immobilizing single stranded nucleic acids from the test medium.

29. The reagent system of Claim 24 wherein the probe is immobilized on a solid support.

30. The reagent system of Claim 24 wherein the probe comprises a binding site for a binding substance and the reagent system additionally comprises an immobilized form of such binding substance.

31. The reagent system of Claim 30 wherein the probe comprises a biotin or hapten moiety and the immobilized binding substance is avidin or an anti-hapten antibody, respectively.

32. The reagent system of Claim 24 wherein the intercalator is a separate, free compound, substantially uncomplexed with nucleic acids.

33. The reagent system of Claim 32 wherein the probe additionally comprises at least one double stranded region.

34. The reagent system of Claim 24 wherein the intercalator is chemically linked to a single stranded region of the probe such that duplex formation in such region results in the formation of intercalation complexes.

35. The reagent system of Claim 24 for use in a sandwich hybridization format which comprises a second nucleic acid probe, the first and second probes respectively comprising at least one single stranded base sequence which is substantially complementary to a mutually exclusive portion of the sequence to be detected.

36. The reagent system of Claim 35 wherein one of the probes is labeled with a detectable chemical group and the other is immobilized.

37. The reagent system of Claim 35 wherein one of the probes is labeled with a detectable chemical group and the other comprises a binding site for a binding substance and the reagent system additionally comprises an immobilized form of such binding substance.

38. The reagent system of Claim 24 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.

39. The reagent system of Claim 24 which additionally comprises a denaturation agent capable of converting double stranded nucleic acids in a test sample into single stranded form.

40. A method for detecting double stranded nucleic acid in a liquid medium, comprising the steps of:
(a) adding to said medium (i) a nucleic acid intercalator and (ii) an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and (b) detecting the binding of said antibody or fragment thereof to said complex.

41. The method of Claim 40 wherein the antibody or fragment thereof is labeled with a detectable chemical group.

42. The method of Claim 41 wherein the detectable chemical group is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

43. The method of Claim 40 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.